臺灣博碩士論文加值系統

本研究的對象是由林口長庚紀念醫院偽菌血症病人血液檢體中分離出來之34株桿菌屬臨床分離菌株，主要研究為菌種分類、抗真菌作用、抗真菌基因分析與抗真菌成份之探討。菌種分類利用生化鑑定（觸媒反應、硝酸鹽還原反應、溶血性試驗、吲朵試驗、檸檬酸試驗、尿素水解作用、歐普氏試驗）及進行分子鑑定（使用聚合酶鏈鎖反應，Polymerase chain reaction; PCR），將34株臨床分離菌株區分為8類：Bacillus cereus、B. thuringiensis、B. coagulans、B. licheniformis、B. pumilus、B. megaterium、B. circulans和B. firmus。在抗真菌作用之分析，發現B. thuringiensis CG 2、B. thuringiensis CG 4、B. pumilus CG 12、B. cereus CG 15、B. cereus CG 20和B. cereus CG 26有明顯抗真菌之作用；其中以對Paecilomyces variotii Tü137之作用最顯著，因此以此6株菌株作進一步抗真菌作用機制之分析。在抗真菌基因之分析，以豐原素之fenB序列設計三組引子，利用PCR來分析34株桿菌屬臨床分離菌株是否含fenB片段。結果在fenB-1及fenB-2這兩組引子增幅出的片段，並非fenB片段；但是標準菌株（B. subtilis F29-3）增幅出的片段為fenB片段。而fenB-2這一組引子，在有明顯抗真菌作用之6株菌中，皆另增幅出1.0 Kb左右的片段。此PCR產物定序分析結果為ilvD而非fenB之基因片段，此基因產物為dihydroxyacid dehydratase，使dihydroxyisovalerate轉化為ketoisovalerate，然而此基因是否與抗真菌作用有相關仍需進一步實驗證明。而抗真菌成份之探討，則將這6株抗真菌臨床分離菌株及枯草桿菌F29-3之醱酵液萃取分離，希望能找出跟抗真菌作用絕對相關之活性成份。亦將細菌與真菌共同培養，結果發現抗真菌物質並非在真菌誘導之環境下產生的，需要有菌體存在一直持續分泌抗真菌物質，才能產生抗真菌之作用。

The object of this study is to examine 34 clinical isolates of Bacillus spp. from blood of pseudo bacteremia patients in Linkou Chang Gung Memorial Hospital, focusing on species classification, antifungal activity, and identification of antifungal gene and active components. Bacterial classification was based on phenotyping (catalase test, reduction of nitrate, hemolysis test, indole production test, citrate utilization test, urease test, Voges-Proskauer test) and genotyping (polymerase chain reaction). All 34 clinical isolates of Bacillus spp. belonged to 8 species: B. cereus, B. thuringiensis, B. coagulans, B. licheniformis, B. pumilus, B. megaterium, B. circulans and B. firmus. From the results of antifungal analysis, B. thuringiensis CG 2, B. thuringiensis CG 4, B. pumilus CG 12, B. cereus CG 15, B. cereus CG 20 and B. cereus CG 26 exhibited significant antifungal activities. These six isolates were further analysed for their antifungal mechanism on Paecilomyces variotii Tü137. It’s well known that Bacillus subtilis F29-3 produces one antibiotic--fengycin, which effectively inhibits the growth of filamentous fungi. According to the sequence of fenB, 3 PCR primer sets were designed to analyze whether any of these 34 clinical isolates of Bacillus spp. contained fenB gene of fengycin. With one primer set, a 1.0 Kb fragment can be amplified in all 6 isolates with antifungal activity. The sequence of the PCR product showed high homology to ilvD. Whether ilvD is related to the antifungal activity remained to be elucidated. Furthermore, to identify the active components contributing to the antifungal activity, the fermentation supernatant were obtained from 6 isolates with antifungal activity and B. subtilis F29-3. After filtration, the supernatants showed no antifungal activity to Paecilomyces variotii Tü137. It appeared that only viable bacteria expressed antifungal activity.

簽名頁
授權書 iii
中文摘要 iv
英文摘要 vi
誌謝 viii
目錄 ix
圖目錄 xii
表目錄 xiv

第一章 緒論 1
1.1 Bacillus spp.之特性 1
1.2 真菌之特性 4
1.3 抗真菌劑（antifungal drug） 6
1.4 菌種鑑定之方法 8
1.5 研究目的 15
第二章 實驗材料與方法 16
2.1 實驗材料 16
2.1.1 菌種 16
2.1.2 培養基、試劑 16
2.2 實驗方法 16
2.2.1 鏡檢 16
2.2.2 生化鑑定 17
2.2.3 分子鑑定 19
2.2.4 抗真菌作用之測試 19
2.2.5 基因組DNA之純化 20
2.2.6 聚合酶鏈鎖反應 21
2.2.7 從瓊脂凝膠中回收DNA片段（Recovering of digested DNA fragment from agarose gel） 22
2.2.8 定序分析 22
2.2.9 細菌醱酵液抗真菌作用之測試 23
2.2.10 真菌與細菌共同培養之抗真菌作用測試 24
第三章 結果與討論 27
3.1 Bacillus spp.之鑑定結果 27
3.2 真菌孢子濃度之測定 29
3.3 抗真菌作用測試之結果 30
3.4 Bacillus spp.與fenB之關係 32
3.5 抗真菌成份之探討 33
3.6 細菌與真菌共同培養之探討 34
第四章 結論 36
參考文獻 72
附錄一、菌種 77
附錄二、培養基、試劑 78
附錄三、本實驗中聚合酶鏈鎖反應（16S rDNA之HV region、rpoB）所用的引子及其鹼基序列 82
附錄四、聚合酶鏈鎖反應（引子：16S rDNA之HV region、rpoB）之反應條件（1）及反應混合液（2） 83
附錄五、本實驗中聚合酶鏈鎖反應（fenB）所用引子之設計
84
附錄六、本實驗中聚合酶鏈鎖反應（fenB）所用的引子及其鹼基序列 85
附錄七、聚合酶錄鎖反應（引子：fenB）之反應條件（1）及反應混合液（2） 86

圖 目 錄
頁次
圖1-1 吲朵試驗反應式之一 10
圖1-2 吲朵試驗反應式之二 11
圖1-3 檸檬酸試驗之反應式 11
圖1-4 VP test作用之反應式 13
圖一、以rpoB為依據之34株桿菌屬臨床分離菌株演化樹狀圖
40
圖二、34株桿菌屬臨床分離菌株細胞於顯微鏡下放大1,000倍所看到的形態 41
圖三、Bacillus spp.抗真菌Penicillium spp.的測試 47
圖四、Bacillus spp.抗真菌Aspergillus versicolor的測試 48
圖五、Bacillus spp.抗真菌Trichophyton rubrum的測試 49
圖六、Bacillus spp.抗真菌Fusarium spp.的測試 50
圖七、Bacillus spp.抗真菌Paecilomyces variotii Tü137的測試
51
圖八、Bacillus thuringiensis CG 2、B. thuringiensis CG 4、B. pumilus CG 12、B. cereus CG 15、B. cereus CG 20、B. cereus CG 26及枯草桿菌F29-3抗真菌的測試 52
圖九、Bacillus thuringiensis CG 2、B. thuringiensis CG 4、B. pumilus CG 12、B. cereus CG 15、B. cereus CG 20、B. cereus CG 26及枯草桿菌F29-3培養七天之菌液的抗真菌測試 54
圖十、Bacillus thuringiensis CG 2、B. thuringiensis CG 4、B. pumilus CG 12、B. cereus CG 15、B. cereus CG 20、B. cereus CG 26及枯草桿菌F29-3培養七天之醱酵液的抗真菌測試 56
圖十一、Bacillus thuringiensis CG 2、B. thuringiensis CG 4、B. pumilus CG 12、B. cereus CG 15、B. cereus CG 20、B. cereus CG 26及枯草桿菌F29-3與真菌不同濃度比（細菌：真菌孢子液＝10：1）共同培養五天之菌液的抗真菌測試 58
圖十二、Bacillus thuringiensis CG 2、B. thuringiensis CG 4、B. pumilus CG 12、B. cereus CG 15、B. cereus CG 20、B. cereus CG 26及枯草桿菌F29-3與真菌不同濃度比（細菌：真菌孢子液＝10：1）共同培養五天之醱酵液的抗真菌測試 60

表 目 錄
頁次
表一、34株革蘭氏陽性菌臨床分離菌株之生化鑑定 62
表二、34株革蘭氏陽性菌臨床分離菌株之菌種鑑定推測結果 64
表三、34株革蘭氏陽性菌臨床分離菌株對五株真菌之抑制作用 65
表四、Bacillus thuringiensis CG 2、B. thuringiensis CG 4、B. pumilus CG 12、B. pumilus CG 14、B. cereus CG 15、B. cereus CG 20及B. tubtilis F29-3抗真菌能力之比較 67
表五、Bacillus thuringiensis CG 2、B. thuringiensis CG 4、B. pumilus CG 12、B. cereus CG 15、B. cereus CG 20、B. cereus CG 26及B. subtilis F29-3培養不同天數之抗真菌（Paecilomyces variotii Tü137）能力比較 68
表六、（A）Bacillus thuringiensis CG 2、B. thuringiensis CG 4、B. pumilus CG 12、B. cereus CG 15、B. cereus CG 20、B. cereus CG 26及B. subtilis F29-3與真菌（Paecilomyces variotii Tü137）共同培養不同天數與不同濃度比之抗真菌（Paecilomyces variotii Tü137）能力比較 69
表六、（B）Bacillus thuringiensis CG 2、B. thuringiensis CG 4、B. pumilus CG 12、B. cereus CG 15、B. cereus CG 20、B. cereus CG 26及B. subtilis F29-3與真菌（Paecilomyces variotii Tü137）共同培養不同天數與不同濃度比之抗真菌（Paecilomyces variotii Tü137）能力比較 70
表六、（C）Bacillus thuringiensis CG 2、B. thuringiensis CG 4、B. pumilus CG 12、B. cereus CG 15、B. cereus CG 20、B. cereus CG 26及B. subtilis F29-3與真菌（Paecilomyces variotii Tü137）共同培養不同天數與不同濃度比之抗真菌（Paecilomyces variotii Tü137）能力比 71

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